In an important type of in situ assay, a cell (or virus) is incubated with a target-specific probe carrying a reporter group. If the probe binds to the cell or virus, its presence can be detected by a process designed to detect the reporter group. Reporter groups include fluorescent moieties and moieties that participate in chemiluminescent reactions. Such assays are useful to detect viral nucleic acids, human genes of interest, specific cellular antigens, and other biologically important molecules.
A common limitation on the sensitivity that can be achieved with such assays is that the probes can bind to entities other than the target molecule of interest. Those entities will normally be in the target biological entity (cell or virus), but may also be on the solid support on which the biological entity or purified target molecules are immobilized. The present invention involves incubating the probes with an excess of an analogue of the reporter group, the analogue being sufficiently similar to the reporter group so that it will compete for the non-specific binding sites, but sufficiently different from the reporter group that it will not enter into the reaction used to detect the reporter group. In particular, the invention focuses on reporter groups that comprise an aromatic component.
The present invention involves the discovery of compounds useful as background reducers in in situ assays.
In addition to the general benefit of increased assay sensitivity that the background reducers provide, there is the additional advantage that, for a given level of sensitivity to be reached, cell treatment procedures such as cell fixation need not be as detrimental to cell integrity as would be the case without the use of the background reducers. As a result, the cells retain their structural integrity for a longer time, a consideration particularly important for flow cytometry.
Haase has reported that "Aurintricarboxylic acid [`ATCA`] a potent inhibitor of protein-nucleic acid interactions [R. G. Gonzalez, et al., Biochim. Biophys. Acta 562:534-545, 1979], also improves backpounds [Turtinen and Weitgrefe, unpublished]." Ashley T. Haase, in K. L. Valentino, et al., eds., In Situ Hybridization: Applications to Neurobiology, New York: Oxford University Press, 1987. In accordance with the present invention, applicants regard ATCA as an analogue to FITC. Applicants have surprisingly found that in competition with FITC, ATCA reduces background in their invention, apart from any effect on protein-nucleic acid interactions.